Animal models of Autoimmune disease

[Strengths and limitations of animal models of Autoimmune disease]

Abstract

Animal models of autoimmune disease have been of vital importance in the advancement of our understanding of central pathological processes that underlie disease causation. Despite continuing debate over the ethics of using animal models in research, progress in the field has been phenomenal in recent times, and a whole host of models, particularly mouse models, have been developed. In this review we examine the strengths and limitations of the various animal models currently in use to emulate some important autoimmune diseases (rheumatoid arthritis, multiple sclerosis, type 1 diabetes and asthma) and look at the future directions that research in this area may take.

Introduction

In the present paper, the literature on current animal models of selected, clinically important, immunological diseases is appraised. In each case an overview of the up to date experimental evidence is examined for positive and negative aspects of the models in widespread use, with major focus on models that attempt to closely mimic crucial effector mechanisms involved in disease processes to facilitate understanding and additionally to test therapeutic interventions. Where appropriate, models emphasising more upstream mechanisms such as genetic factors and/or specific (isolated) immune processes are also explored.

Murine models have been the most extensively used in immunology research as the species, until recently (Abbott 2004), have been the only organisms with sufficient genetic similarity to humans (the mouse was only the second organism after humans to have its genome sequence determined, the rat being the third) (E S Lander et al. 2001) (Robert H Waterston et al. 2002) (Richard A Gibbs et al. 2004) that are amenable to gene cloning and transgenic methods. This enables investigators to conduct highly controlled in vivo experiments that can be used effectively to draw conclusions about causal relationships that may exist between almost any molecule and the disease process being studied.

The vast number of murine models available encompasses a wide variety of immune disorders - from rheumatoid arthritis to multiple sclerosis. To overcome the difficulty of organising and accessing the large amounts of information on the various models, a large consortium of researchers set up a searchable database - the MUGEN Mouse Database (MMdb) (Aidinis et al. 2007) that allows any researcher to view details about the various models that have been generated. This appears to be the best solution that enables researches to better integrate their investigative efforts, and should be applied to the new rat models that are beginning to become widespread.

Rheumatoid arthritis

Overview

Rheumatoid arthritis (RA) is a common (prevalence = approximately 1%) (Lawrence et al. 1998) systemic disorder the hallmarks of which are chronic, symmetrical, inflammatory synovitis of mainly peripheral joints. Its course is extremely variable and it is associated with nonarticular features that can sometimes be prominent. Over the course of the disease, chronic synovitis (inflammation of the synovial lining of joints, tendon sheaths or bursae with a infiltration of macrophages, T-cells, polymorphs, etc.) leads to erosions of normal articular tissue. These pathological changes manifest clinically as a variety of deformities and contribute to long-term disability. Mortality rates in RA patients are also higher, compared with rates observed in the general population (Lawrence et al. 1998).

Examination of RA tissue specimens from arthroscopy and joint replacement procedures, and other studies in humans suggest that many facets of the innate and adaptive immune systems are involved. Some of the well-established factors involved in RA pathogenesis include genetic susceptibility, abnormal T-cell and subsequently B-cell responses to autoantigens (Imboden 2009). Environmental influences also have a role in disease causation, but these are largely unknown, and many believe that a sensitisation event coupled with abnormal antigen presentation leads to autoimmunity.

Present Models

Earlier animal models, such as collagen-induced (Trentham, AS Townes, and Kang 1977) (Courtenay et al. 1980) (Holmdahl et al. 1990) or proteoglycan -induced arthritis (CIA/PIA) (Glant, Finnegan, and Mikecz 2003), were based on the hypothesis that RA results from an adaptive immune response to a joint-specific antigen. Genetically susceptible mice (and later monkeys (Yoo et al. 1988)) are repeatedly exposed (via intraperitoneal and/or subcutaneous injection) to purified autologous or heterologous type II collagen (or cartilage proteoglycans like aggrecan, in the case of proteoglycan-induced arthritis) (Glant and Mikecz 2004). Further induced models were subsequently generated. This led to the development of a systemic autoimmune arthritis in the subjects. Comprehensive study of these models has yielded vital evidence for the concept that autoimmunity to joint-specific antigens can lead to arthritis.

The experiments on CIA/PIA mice, particularly in the 80s and 90s, were a fruitful model for researchers. The large number of inquiries using the models as a basis clarified the roles that certain cell types (Tada et al. 1996) (Peterman et al. 1993) and molecular mediators such as MHC molecules (PH Wooley et al. 1981), autoantibodies (Jasin 1985) and the complement system (Watson et al. 1986) play in the pathogenesis of arthritis. In addition, much was deduced about genetic predisposition to RA-like illness from study of these models.

More recently, models that exhibit spontaneous onset of arthritis have become available. These provided valuable insights into the role of key molecules in the pathogenesis of arthritis. The TNF-a transgenic mouse was amongst the first of the models that show spontaneous onset of arthritis and the finding that over-production of TNF-α alone is sufficient to induce erosive arthritis led to a fundamental change in the understanding of the disease process. It highlighted the sensitivity and response of sinoviocytes to circulating cytokines, and eventually led to the development of anti-TNFa therapies that have become so essential in the management of RA refractory to standard disease modifying agents (Feldmann and Maini 2001). Other novel models have also led to further clarification of cytokine signalling as possible fundamental events in RA pathogenesis. Atsumi and colleagues generated a mouse line with point mutations in the gp130 (glycoprotein 130) that developed RA-like joint disease (Atsumi et al. 2002).

In addition, multiple strains of T-cell receptor (TCR) transgene models have been produced. One such model expresses an influenza hemagglutinin antigen in combination with a transgene for hemagglutinin-reactive TCR with variable affinity for the antigen(Caton et al. 1991). The mice that expressed the low-affinity TCR unexpectedly developed a symmetric polyarthritis, implying that the T-cells failed negative selection for self-reactivity during maturation in the thymus. Similarly, other TCR transgene models support the role of impaired T-cell maturation as important events that lead to RA-like phenotypes, for example Kouskoff and colleague's KRNxNOD strain developed from crossing a TCR transgenic line with a NOD (nonobese diabetic mouse) strain(Kouskoff et al. 1996). The KRNxNOD mice model demonstrated that systemic self-reactivity can induce joint-specific disease. Pathogenic immunoglobulins (Igs) that recognise the ubiquitous cytoplasmic enzyme glucose-6-phosphate isomerase were identified as the causative factor in the model and transmission of disease was also confirmed(Matsumoto et al. 2002).

The induced models of RA, although invaluable for their contribution to the understanding of the effector mechanisms important in RA pathogenesis, do not adequately allow for study of upstream mechanisms. Moreover, even though many examples of successful therapeutic trials against molecular targets in mice exist (Mikecz et al. 1995) (Kakimoto et al. 1992) (Miyahara et al. 1993) and some of this success translated into success in human cell line studies, this has not as of yet resulted in the development of clinically useful treatments. (Unlike the development of anti-TNF- α therapy that resulted from the study of spontaneous models) This narrowed view of disease pathogenesis that induced models provide was revealed after the development of spontaneous models of RA that emphasise the significance of synoviocyte sensitivity to cytokines as fundamental events in disease causation. Logically it follows on from this that further development of original models must occur in order to piece together the glimpses of overall disease causation that different models give us.

Multiple Sclerosis

Overview

Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system. Overall, in Europe and North America the annual incidence is 2-10 per 100 000, making it the most common neurological disease in young adults. The disease is characteristically results in the formation of multiple plaques of demyelination within the brain and spinal cord. Plaques are “disseminated in time and place”, hence the old name disseminated sclerosis. The clinical, pathological and immunological phenotype of MS is unpredictable, and there is wide variation in severity. Clinically patients are classically divided into two categories those that present with relapsing-remitting disease, or with steady (primary) progressive neurological disability. The subsequent course of disease is unpredictable, although most patients with a relapsing-remitting disease eventually develop secondary progressive disease. Pathological examination of CNS tissue from MS patients with acute relapsing-remitting disease typically reveals plaques of focal demyelination in perivenular white matter, as opposed to the more widespread involvement that is more typical of primary progressive MS (Compston et al. 2005). In addition, there is a high inter-patient inconsistency in the extent of axonal damage as well as remyelination and repair suggesting that multiple factors may play an important role in the pathogenesis of MS (H Lassmann, W Brück, and C Lucchinetti 2001).

Present Models

The experimental autoimmune encephalomyelitis (EAE) models of MS, originally identified in monkeys when studying the cause of encephalomyelitis developing post-inoculation with Pasteur rabies vaccine (Rivers and Schwentker 1935), have been extensively studied and reviewed. Since then EAE was demonstrated in many different species (including guinea pigs and rats) and from these studies it became clear that EAE can reproduce many of the clinical, neuropathological and immunological aspects of multiple sclerosis and other demyelinating pathologies like acute disseminated encephalomyelitis (ADEM) (Hohlfeld and Wekerle 2001). Since their development, mouse models began to gain popularity due to the provision of producing genetically modified strains, which greatly enhances the chances of proving key molecules' roles in disease pathogenesis.

There is some variation in the development of the resultant pathology (Huseby et al. 2001) depending on which combination of antigen and adjuvant is used, for example, demyelination MOG-induced EAE is exacerbated by simultaneous administration of anti-MOG compared to PLP/MBP-induced EAE (Sun et al. 2001). This is one of the criticisms of the EAE model. However, some researchers look at this heterogeneity as an advantage and instead of argue that the variety of models available allows the dissection of the roles of key cells and molecular mediators in the disease process, and that this may reflect the complexity of MS as a clinical syndrome. There is some support for this view from studies in humans that prove that multiple routes to MS are possible, and that there is considerable inter-patient and even intrapatient variation in which molecular pathways are operating (Claudia Lucchinetti et al. 2000) (Furlan et al. 1999). Moreover, the development of newer models, even models that mimic relapsing remitting disease (such as the Congenic Lewis strains), has led to the realisation that improvements in molecular biology techniques will pave the way for more pertinent models in the future.

The most discouraging aspect of EAE as a model is the fact that so far the vast majority of therapeutic approaches that were successful in model organisms have not been shown to be efficacious in humans. In fact, from the multitude of substances tested, glatiramer acetate represents the only drug in clinical use today that was first identified from successful trials in EAE models (Lisak et al. 1983) (Munari, L, Lovati, Roberta, and Boiko, Alexei 2003).

Type I Diabetes

Overview

Type 1 diabetes (T1D) belongs to a family of HLA-associated immune-mediated organ-specific diseases. T-cell entry into pancreatic islets and destruction of insulin-producing ß-cells is thought to be central to the pathogenesis. Support for the immunological basis of the disease includes the accumulation of islet antibodies (including those specific to insulin itself, the enzyme glutamic acid decarboxylase (GAD), and the intracellular portion of two islet peptides from the tyrosine phosphatase family) with differential specificities for beta-cell proteins in patients' serum present in some 90% of newly presenting patients (Kumar and Clark 2005). In addition, epidemiological evidence suggests associations with certain HLA-haplotypes (notably HLA-DR3-DQ2 and HLA-DR4-DQ8) and other organ-specific autoimmune diseases including autoimmune thyroid disease, Addison's disease and pernicious anaemia. Furthermore, systemic immunosuppressive treatment following diagnosis, for example with ciclosporin or autologous nonmyeloablative hematopoietic stem cell transplantation, has been shown to prolong beta-cell survival and cause clinically significant regression of disease (Voltarelli et al. 2007).

The involvement of T-cell activation in the pathogenesis of T1D is thought to be vitally important, as evidenced by the recent success of anti-CD3 antibodies (e.g. teplizumab/MGA031) in clinical trials(Herold et al. 2002) (Keymeulen et al. 2005).

Present Models

Similar to other immune diseases, mice are most extensively used to model T1D. The nonobese diabetic (NOD) mouse strain (Makino et al. 1980) that exhibits highly penetrant, rapid-onset disease is the archetypal (some may even say overused) T1D model. The combination of a specific MHC haplotype and a large set of susceptibility-related genes (many that are shared with humans) produces the NOD phenotype, which can be prevented or reversed by a vast number of tested interventions (Yamada et al. 1982) (Shoda et al. 2005).

Sets of spontaneous mouse models of T1D that result from specific known mutations or pathway defects are also available. These include animals with particular defects in such fundamental genes as those encoding the transcription factor Foxp3 (Wan and Flavell 2007) (Appleby and Ramsdell 2008), interleukin 2, the kinase (Glant and Mikecz 2004)Zap70, Fas and other important immune mediators that are thought to be important in self-tolerance mechanisms.

Humanised models of T1D (similar to those in use for RA) have been produced. Transgenic NOD mice expressing human MHC molecules (for example HLA-A2) as well as particular murine MHC class I molecules have been used to study responses of islet-infiltrating lymphocytes to a collection of various known antigens (Serreze, Marron, and Dilorenzo 2007) (Enée et al. 2008). Innovative approaches to identifying T-cell subpopulations and antigens that drive the T1D disease process have been employed by some investigators in order to answer the question of whether there is a particular TCR that causes the demise of most beta cells or at least commonly triggers the process. Burton and others used retroviral mediated stem cell gene transfer techniques to produce multiple strains of mice each with a monoclonal population of T-cells expressing particular TCRs for known antigens, some of which developed T1D-like illness. They went on to show that as few as 1,000 of such cells are capable of causing rapid development of diabetes following transfer into NOD/SCID (Severe combined immunodeficiency) mice (Amanda R. Burton et al. 2008). However, the technology currently available does not allow easy and reliable detection of such cells in very low numbers in blood, and such assocations cannot be proven in humans without great difficulty.

NOD strains do have multiple drawbacks. First, the fact that the NOD mouse was developed with a unique set of genotypic circumstances that is unlikely to exist in a substantial fraction of the human population, if it exists at all, means that the model obviously overemphasises the genetic factors that lead to T1D. This is important, as T1D has been shown in twin and adoption studies to have less of a genetic component than T2D (approx 65% vs. 90% monozygotic twin concordance rates (Redondo et al. 2008)), which in itself is also thought to be a complex genetic disorder with multiple small-effect genes combined with environmental factors eventually leading to disease. Thus, if insights into environmental triggers for T1D are to be gained, NOD mice provide an insufficiently broad perspective.

Asthma

Overview

Asthma is a common chronic inflammatory condition of the lung airways whose cause is incompletely understood. Worldwide prevalence of asthma is increasing, and varies from region to region with highest rates of up to 30% (e.g. in Ireland and Australia) (Beasley et al. 2000) underlying the need to understand the disease process better. Symptoms are cough, paroxysmal wheeze, chest tightness and episodic shortness of breath, often worse at night. It has three characteristic features: airflow limitationwhich is usually reversible spontaneously or with treatment, airway hyperresponsivenessto a wide range of stimuli and inflammation of the bronchiwith eosinophils, T-cells and mast cells with associated plasma exudation, oedema, marked smooth muscle hypertrophy, mucus plugging and epithelial damage.

Present Models

Producing models for asthma has been a challenge and most research has concentrated on methods to give rise to allergic inflammation by sensitizing (for instance by repeated intraperitoneal injection) and challenging the animals' airways with a variety of foreign proteins (by inhalation of aerosol) (J.C. Kips et al. 2003) (Bates, Rincon, and Irvin 2009). This is because specific aetiological factors for the disease in humans are yet to be determined, and diagnosis relies on clinical judgement, rather than objective testing. Thus the focus is on developing organisms that exhibit phenotypic characteristics similar to those are observed in human disease, particularly acute allergic inflammation, hyperresponsiveness and pathological changes such as airway remodelling and eosinophil infiltration.

Approaches taken include using ovalbumin (Tormanen et al. 2005) and other antigens. These models show elevated serum IgE concentrations, eosinophil activation, and a Th2-like T-cell response with increased levels of interleukin (IL)-4, IL-5, IL-13, eotaxin, and RANTES (S P Hogan et al. 1997) (Bell et al. 1996)(Hamelmann et al. 1997). Furthermore, eversible airway obstruction is also a feature of this model. Findings such as this provided evidence for the classical Th2-response-driven asthma pathway. This, however, is an oversimplification and support for the involvement of Th1 cells that positively regulate the Th2 response also exists (Riese, Finn, and Shapiro 2004) (Dahl et al. 2004).

Following on from the development of these models were studies in knockout mice some of which seemed to identify promising therapeutic prospects. One such example is that of the IL-5 -/- mice which were shown in meticulous experiments to be protected from both airway hyperresponsiveness (PS Foster et al. 1996), and chronic airway remodelling (Cho et al. 2004) when subjected to the ovalbumin inhalation. This led to the development of anti-IL-5 therapies that were unfortunately unsuccessful in clinical trials for both mild acute asthma (Leckie et al. 2000) and chronic severe asthma (Johan C. Kips et al. 2003). Nonetheless, deeper investigation of the role of eosinophils (which are activated by IL-5) in airway remodelling showed that eosinophils are only crucial in more chronic aspects such as the deposition of collagen and smooth muscle cell proliferation (Humbles et al. 2004). This implies that, as can be inferred from the animal model experiments, that IL-5 may be important in the early stages of disease development, and thus anti-IL-5 therapy may only be efficacious in the prevention of asthma, an effect that may not be clinically significant.

Although many have reported findings in mice that very closely emulate human asthma, many important limitations to the interpretation of these findings exist. First, many of the experiments demonstrating pathological findings in the models similar to those found in asthma (for example Hogan, Ramsey and others' ovalbumin-challenged model) obtained tissues for histological examination days after cycles of ovalbumin inhalation, have been challenged by conflicting data. Some investigators (Erjefält et al. 1998) have described the regression of some of the changes (namely increased microvascular permeability and extravasation of plasma) within minutes of ovalbumin challenges with no observable late-phase changes. This is not reflected in some of the more publicised findings, in which most investigators report finding changes in tissues obtained even days after challenges. In addition, the models also differ qualitatively in how they exhibit some changes that are observed. Most of the papers documenting increased vascular permeability, did not actually demonstrate plasma extravasation into the epithelial lining or the airway lumens.

CONCLUSIONS

The ultimate goal of pursuing the study of animal models is the translation of expertise and knowledge into clinically useful diagnostic and therapeutic strategies. Therefore, if one were to be absolutely critical of a particular model in use, the emphasis should perhaps be on its usefulness in achieving these goals, rather than on correlating specific pathological and/or mechanistic characteristics of the model with features of human disease.

A common theme that emerges when looking at animal models in widespread use in immunology is the need for novel strains that enable study of upstream mechanisms (as effector mechanisms are now vastly better understood) in disease reflected in the recent departure from induced-models towards more spontaneous models that can be observed when scrutinising the literature. This predictably involves bringing together knowledge from studies involving human tissue and it is now clear that in order to overcome the inevitable limitations of modelling, researchers must look for novel ways to incorporate knowledge gained from, for example linkage and (genome wide) association studies, into the design of animal models. Many such examples already exist, as illustrated by the development of the transgenic mice expressing human TNF-a to serve as a predictive genetic model of arthritis (Keffer et al. 1991). This is also a commonly described downfall of the NOD strain of mice in use for the study of T1D, which is criticised for its indirect approach towards studying disease susceptibility genes. Many authors (von Herrath and Nepom 2009) believe that “instead of looking for homologues in humans of the NOD autoimmunity-related genes, variations and homologues of the human diabetes susceptibility-related alleles identified should be introduced into mouse strains that carry the NOD H-2g7 MHC but that lack the NOD background autoimmunity-related genes”. This would lead to identification of key molecules in the initiation and progression of T1D and subsequent detection of potentially clinically useful biomarkers (for example autoantibodies, serum cytokines). Similarly, if this formula is applied to any immunological disease, and the joint use of results from human and animal models finally harnesses the capability of today's technology, we may see a burst of therapeutic and diagnostic leads emerging in the post-genomic era.

Another future direction that can be perceived in the development of animal models of autoimmune disease (much the same as for other diseases) is the increase in popularity of the rat model. The successful cloning of rats (Zhou et al. 2003) and the sequencing of the Brown Norway rat genome (Richard A Gibbs et al. 2004) followed by the recent use of zinc-finger nuclease technology in the generation of knockout mice (Geurts et al. 2009) has revitalised the interest in the rat model. This will undoubtedly lead to the development of better models of disease, as the rat is closer to humans (Richard A Gibbs et al. 2004) than any other organism amenable to such manipulation at the molecular level.

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